BRET Sensors for Imaging Membrane Integrity of Microfluidically Generated Extracellular Vesicles
Ramasamy Paulmurugan1,2, Yi Liu3,4, Uday Kumar Sukumar3,4
1Molecular Imaging Program at Stanford, Stanford University School of Medicine, Palo Alto, CA, USA. paulmur8@stanford.edu.
Methods in Molecular Biology (Clifton, N.J.)
|July 14, 2022
Summary
We developed a novel microfluidic method to create uniform, microRNA-loaded extracellular vesicles (EVs) for cancer therapy. A new bioluminescence sensor system was used to confirm the integrity of these reconstructed EVs.
Area of Science:
- Biotechnology
- Nanomedicine
- Cancer Therapy
Background:
- Extracellular vesicles (EVs) are natural nanodelivery vehicles for therapeutics, particularly in cancer treatment.
- Existing methods for generating engineered EVs face challenges in uniformity and loading efficiency.
- Novel strategies are needed to ensure the functional integrity of engineered EVs for in vivo applications.
Purpose of the Study:
- To develop a microfluidic-based strategy for generating uniform, microRNA-loaded reconstructed extracellular vesicles (EVs) for cancer therapy.
- To introduce a novel bioluminescence resonance energy transfer (BRET) sensor system for evaluating the membrane integrity of reconstructed EVs.
- To demonstrate the utility of this system for in vivo imaging and functional assessment of engineered EVs.
Main Methods:
- Utilized a microfluidic system to reconstruct vesicles using isolated EVs and cell membranes from various sources.
- Engineered cells to express a membrane-insertable BRET sensor (GAP43-derived palmitoylation signal peptide) for vesicle tracking.
- Isolated EVs from engineered cells, processed them using microfluidics, and evaluated membrane integrity via BRET imaging.
Main Results:
- The microfluidic system produced reconstructed vesicles with uniform sizes and high microRNA loading efficiency.
- Loading efficiency was independent of the source of the input membrane (EVs or cell membranes).
- The BRET sensor system successfully enabled optical imaging and evaluation of the membrane integrity of reconstructed EVs.
Conclusions:
- Microfluidic reconstruction offers a robust method for generating engineered extracellular vesicles (EVs) for cancer therapy.
- The developed BRET sensor system provides a valuable tool for assessing the functional integrity of reconstructed EVs.
- This combined approach holds significant promise for advancing nanodelivery systems in cancer treatment.
